The addition of bisecting N-acetylglucosamine residues to E-cadherin down-regulates the tyrosine phosphorylation of beta-catenin

The enzyme GnT-III (beta 1,4-N-acetylglucosaminyltransferase III) catalyzes the addition of a bisecting N-acetylglucosamine (GlcNAc) residue on glycoproteins. Our previous study described that the transfection of GnT-lll into mouse melanoma cells results in the enhanced expression of E-cadherin, which in turn leads to the suppression of lung metastasis. It has recently been proposed that the phosphorylation of a tyrosine residue of beta-catenin is associated with cell migration. The present study reports on the importance of bisecting GlcNAc residues by GnT-lll on tyrosine phosphorylation of beta-catenin using three types of cancer cell lines. An addition of bisecting GlcNAc residues to E-cadherin leads to an alteration in cell morphology and the localization of beta-catenin after epidermal growth factor stimulation. These changes are the result of a down-regulation in the tyrosine phosphorylation of beta-catenin. In addition, tyrosine phosphorylation of beta-catenin by transfection of constitutively active c-src was suppressed in GnT-III transfectants as well as in the case of epidermal growth factor stimulation. Treatment with tunicamycin abolished any differences in beta-catenin phosphorylation for the mock vis à vis the GnT-lll transfectants. Thus, the addition of a specific N-glycan structure, the bisecting GlcNAc to E-cadherin-beta-catenin complex, down-regulates the intracellular signaling pathway, suggesting its implication in cell motility and the suppression of cancer metastasis.     

Chemical modulators of autophagy as biological probes and potential therapeutics

Autophagy is an evolutionarily conserved mechanism for protein degradation that is critical for the maintenance of homeostasis in man. Autophagy has unexpected pleiotropic functions that favor survival of the cell, including nutrient supply under starvation, cleaning of the cellular interior, defense against infection and antigen presentation. Moreover, defective autophagy is associated with a diverse range of disease states, including neurodegeneration, cancer and Crohn's disease. Here we discuss the roles of mammalian autophagy in health and disease and highlight recent advances in pharmacological manipulation of autophagic pathways as a therapeutic strategy for a variety of pathological conditions.     

Salinity Decreases Nitrite Reductase Gene Diversity in Denitrifying Bacteria of Wastewater Treatment Systems

Investigation of the diversity of nirK and nirS in denitrifying bacteria revealed that salinity decreased the diversity in a nitrate-containing saline wastewater treatment system. The predominant nirS clone was related to nirS derived from marine bacteria, and the predominant nirK clone was related to nirK of the genus Alcaligenes.     

Dopaminergic neuronal loss in transgenic mice expressing the Parkinson's disease-associated UCH-L1 I93M mutant

The I93M mutation in ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) was reported in one German family with autosomal dominant Parkinson's disease (PD). The causative role of the mutation has, however, been questioned. We generated transgenic (Tg) mice carrying human UCHL1 under control of the PDGF-B promoter; two independent lines were generated with the I93M mutation (a high- and low-expressing line) and one line with wild-type human UCH-L1. We found a significant reduction in the dopaminergic neurons in the substantia nigra and the dopamine content in the striatum in the high-expressing I93M Tg mice as compared with non-Tg mice at 20 weeks of age. Although these changes were absent in the low-expressing I93M Tg mice, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment profoundly reduced dopaminergic neurons in this line as compared with wild-type Tg or non-Tg mice. Abnormal neuropathologies were also observed, such as silver staining-positive argyrophilic grains in the perikarya of degenerating dopaminergic neurons, in I93M Tg mice. The midbrains of I93M Tg mice contained increased amounts of insoluble UCH-L1 as compared with those of non-Tg mice, perhaps resulting in a toxic gain of function. Collectively, our data represent in vivo evidence that expression of UCHL1(I93M) leads to the degeneration of dopaminergic neurons.     

Fluorescence polarization studies on myosin-substrate interaction

The degree of polarization of the intrinsic fluorescence of purified myosin was estimated. On addition of ATP, polarization of the fluorescence of myosin increased when excited at wavelengths longer than 300 nm. In kinetic studies, coupled with the decay of the increased intensity of fluorescence of myosin, the increased polarization of the fluorescence decreased when the ATP was depleted. The decay of the increased polarization of fluorescence of myosin was specific to MgATP. According to the theory of polarization of the fluorescence of proteins, it is likely that some tryptophan residues of myosin, which are responsible for the increase in the fluorescence intensity and polarization when myosin interacts with substrates, reduce their local freedom of rotation.     

How is autonomic nervous system activity in subjects who are sleepy but are unable to sleep in the daytime?

We investigated changes in the activity of the autonomic nervous system (ANS) in the relaxed condition in subjects who felt sleepy, but were unable to sleep. A total of 1021 subjects underwent daytime polysomnography. The sleep latency (SL) and the visual analog scale (VAS) were used to assess “immediate” objective and subjective sleepiness, respectively. The subjects were assigned to an “Alert-Alert” group (VAS ≤ 25 mm, SL ≥ 8 min), a “Sleepy-Alert” group (VAS ≥ 75 mm, SL ≥ 8 min), or a “Sleepy-Sleepy” group (VAS ≥ 75 mm, SL ≤ 4 min). In order to assess the ANS, the spectral analysis and the geometric method were used. The ANS data collected during the relaxed condition (after lights off, post-LO) was compared to that obtained during the control condition (before lights off, pre-LO). From the spectral analysis, a significant decrease of sympathetic function and an increase of parasympathetic function at post-LO in the Sleepy-Sleepy group, a tendency for sympathetic function decrease at post-LO in the Alert-Alert group, and no significant changes to sympathetic and parasympathetic function in the Sleepy-Alert group were observed. The results from the geometric method supported the results of the spectral analysis in the Alert-Alert group and the Sleepy-Sleepy group. The results of this study suggest that the ANS plays a role in individuals who are unable to sleep even though they feel sleepy and are given the opportunity to sleep.

     

Population genetic structure of Scombrops boops (Percoid, Scombropidae) around the Japanese archipelago inferred from the cytochrome b gene sequence in mitochondrial DNA

The gnomefish (Scombrops boops) is a member of the percoid family Scombropidae, which includes a single genus and three to four species worldwide. Since little is known about the ecology of this species, here, sequencing analysis of the cytochrome b gene (1141 bp) in mitochondrial DNA detected 101 haplotypes from 186 individuals of S. boops collected from waters at seven localities around the Japanese archipelago. A single haplotype (Sb2) was the most abundant in the combined populations of S. boops from various localities. Genetic population structure analyses revealed no significant differences among these populations (Fst = - 0.0313-0.0195; Φst = - 0.0505-0.0615) with high haplotype diversity and low nucleotide diversity. This suggests that S. boops around the Japanese archipelago constitutes a single population, and indicates that the genetic structure of this population may be influenced by larval and egg dispersal in association with warm currents.     

Typical three-domain cry proteins of Bacillus thuringiensis strain A1462 exhibit cytocidal activity on limited human cancer cells

Bacillus thuringiensis strain A1462 produced two parasporal inclusion proteins with a molecular mass of 88 kDa that were converted to 64-kDa toxins when activated by proteinase K digestion. Both toxins exhibited strong cytocidal activity against two human cancer cell lines, HL60 (myeloid leukemia cells) and HepG2 (liver cancer cells), while low or no toxicities were observed against 11 human and three mammalian cell lines, including four non-cancer cell lines. The cytotoxicity of both toxins on susceptible cells was characterized by rapid cell swelling. Gene cloning experiments provided two novel genes encoding 88-kDa Cry proteins, Cry41Aa and Cry41Ab. The amino acid sequences of the two proteins contain five block regions commonly conserved in B. thuringiensis insecticidal Cry proteins. This is the first report of the occurrence of typical three-domain Cry proteins with cytocidal activity preferential for cancer cells.